Biomedical subjects

A Gautreau

Publications and source records attributed to A Gautreau.

4 recordsLinked to original sources

PubMed1998

Simultaneous quantitation of substance P-encoding preprotachykinin alternatively spliced mRNAs and substance P receptor NK-1 mRNA by an RNase protection assay.

Tachykinins form a family of peptides with neurotransmitter/neuromodulator function. Four tachykinins, substance P, neurokinin A, neuropeptide gamma and neuropeptide K, are encoded by the same PreProTachykinin (PPT) gene. Alternatively spliced mRNAs encode different combinations of these peptides (Brown, E.R., Harlan, R.E., Krause, J.E., Gonadal steroid regulation of substance P (SP) and SP-encoding mRNA in the rat anterior pituitary and hypothalamus, Endocrinology, 126 (1990) 330-340; Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881-885). The proportion of PPT mRNAs varies from tissue to tissue (Carter, M.S., Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214), and within the rat hypothalamus according to the estrous cycle-related hormonal status (Gautreau, A., Duval, P., Kerdelhué, B., Variations in substance P-encoding preprotachykinin and substance P receptor NK-1 mRNA transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of beta-preprotachykinin and NK-1 mRNA, Mol. Brain Res., (1997) in press). Tachykinin receptors as well as tachykinins are regulated at the mRNA level. A fully quantitative method is needed to deal with the complex physiological regulation of the tachykinin system. Here, we describe an RNase protection assay that allows the simultaneous quantitation of alternatively spliced PPT mRNAs, Substance P receptor NK-1 mRNA, and glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA as an internal control, in the rat hypothalamus. The advantages of this method are its high sensitivity (0.1 pg) and a wide range of linearity (more than 3 orders of magnitude). Moreover, this protocol provides guidelines to set up a quantitative multiprobe RNase protection assay for other genes.

Alternative Splicing↗

PubMed1997

Variations in levels of substance P-encoding beta-, gamma-preprotachykinin and substance P receptor NK-1 transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of beta-preprotachykinin and NK-1 mRNA.

Using a sensitive RNase protection assay, the simultaneous quantification of hypothalamic beta-, gamma-preprotachykinin (PPT) and SP receptor NK-1 transcripts was performed throughout the estrous cycle. The amounts of these 3 transcripts were up-regulated during diestrus II-proestrus night (2-, 1.5- and 1.3-fold, respectively). These levels returned to low values during the proestrous day in the case of gamma-PPT mRNA and during the estrus-diestrus I night in the cases of beta-PPT and NK-1 mRNAs. These results implicate a differential regulation in amounts of the two alternatively spliced PPT transcripts. The 160 hypothalami of this study had been previously assayed for amounts of substance P (SP) and neurokinin A (NKA) peptides [P. Duval, V. Lenoir, S. Moussaoui, C. Garret and B. Kerdelhué, Substance P and neurokinin A variations throughout the rat estrous cycle; comparison with ovariectomized and male rats: I. Plasma, hypothalamus, anterior and posterior pituitary, J. Neurosci. Res., 45 (1996) 598-609]. Variations in mRNA and peptide levels were compared by statistical analysis. Surprisingly, variations in SP level paralleled those in beta-PPT mRNA level and variations in NKA level paralleled those of gamma-PPT mRNA level, although beta- and gamma-PPT transcripts encode both SP and NKA. Furthermore, the level of NK-1 mRNA was positively correlated with the level of beta-PPT mRNA (r = 0.90, P < 10(-58)) and with the level of SP peptide (r = 0.30, P < 10(-3)) but not with the level of NKA peptide. This analysis suggests that SP receptor NK-1 mRNA could be physiologically regulated by SP peptide in the rat hypothalamus.

Analysis of Variance↗

PubMed1997

ActA is a dimer.

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Bacterial Proteins↗

PubMed1997

Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells.

The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane-cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.

Animals↗